Use of biomarkers in saliva to evaluate the toxicity of agents and the function of tissues in both biomedical and environmental applications

ABSTRACT

Sensitive methods using a variety of biomarkers in oral saliva of humans and animals to detect, measure, and quantify the presence of infectious and non-infectious agents and the functional status of living tissues in both (1) biomedical and (2) environmental applications. With the in vivo biomedical applications, saliva samples are taken from human or animal subjects and biomarkers, such as enzyme or antibody levels, are measured. The extent of exposure to an agent is measured by the presence of specific chemical or biological constituents, by the degree of the inhibition of enzymes, or by the changes in the amount of the biochemicals in saliva. With the in vitro environmental applications, enzymes or other biochemicals from animal or human saliva can be used to monitor the presence of toxic or reactive agents in tissue samples, urine, feces, milk, air, water, soil, or plants. The amount of toxicant in samples is estimated from a standard curve for that agent.

This application is a continuation of application Ser. No. 08/478,498filed on Jun. 5, 1995, now U.S. Pat. No. 5,686,237.

BACKGROUND OF THE INVENTION

1. Field of the Invention

Generally, the present invention relates to the detection andmeasurement of biomarkers in saliva. More particularly, the subjectinvention describes methods which employ saliva biomarker chemicalspecies from human and animal subjects for detection, quantification,and evaluation of hazardous and toxic chemical or biological agents.

2. Description of the Related Art

The detection and quantification of potentially hazardous or toxicagents and potentially hazardous infectious biological agents aresubjects of concern in biomedical and environmental fields as well as tothe general public. Humans and animals are exposed to a wide variety ofhazardous agents that may or may not be infectious endogenousxenobiotics. For example, on a world wide basis, pesticides have beenestimated to cause as many as 500,000 illnesses annually, with as manyas 20,000 deaths resulting from pesticide exposure. Informationregarding the presence and quantity of hazardous agents within humans oranimals, or in the environment where humans or animals may be exposed,is useful in prescribing medical treatments, avoiding further exposure,and assessing levels of environmental contamination.

Several fields of the biomedical sciences are concerned with thecharacterization and diagnosis of abnormal or pathologic conditionswithin the body of a human or animal. There are many types ofneurological and systemic diseases leading to abnormal levels ofspecific biochemicals within the body. These biochemicals need to bedetected and measured for the purpose of diagnosis. Currently, detectionand measurement methods rely on tissue and blood samples which are usedto monitor exposure to infectious agents or toxicants in human andanimals or the presence of diseases and contaminating, The presentlyused techniques are generally limited to detecting tissue damageresulting from acute exposure to high levels of an agent, toxin,toxicant, and the like or tissue damage caused by advanced diseases orexposure to chemicals. The sensitivity of procedures utilizing blood ortissue samples is limited by many technical constraints which preventdetection at low levels of chemical agent or at early stages ofdiseases. For example, the chronic exposure to low levels of someorganophosphate compounds and organic solvents can cause delayedneuropathy in human and animals, and the resulting changes in thenervous system cannot be monitored by currently used methods whichcharacterize blood enzymes.

Acetylcholinesterase, carboxylase, and other enzymes present in bloodand tissue have been used for many years as biomarkers to monitor thetoxicity of agents both in vivo and in vitro. The presence of toxic orreactive agents in blood or tissue samples can often be detected by thedegree of inhibition of one or more enzymes that are sensitive tobinding and inhibition by those toxic agents.

The present invention introduces the use of human and animal salivabiomarkers for sensitive and non-invasive detection and quantificationof chemical and biological agents, usually hazardous substances,including infectious and non-infectious agents. Several advantages areassociated with the use of biomarker species that are enzymes fromsaliva as disclosed herein instead of from blood or tissue as previouslycarried out. For example, saliva biomarkers are exposed to toxic orreactive agents systemically through the blood and also locally byinhalation and/or ingestion through the mouth, while plasma and tissueenzymes are exposed only by blood uptake. This makes the intensity andthe frequency of saliva exposure to toxicants greater than those inblood and tissues.

Another advantage is that saliva contains 0.2-0.5% protein, versus 7.2%in plasma, and the albumen content of saliva is 1% that of plasma.Chemicals or other hazardous agents may undergo nonspecific binding withvarious plasma proteins due to the high protein concentration, resultingin a reduction in enzyme sensitivity to the effects of the chemicals orother toxic agents. Still another advantage is the relatively smallvolume of saliva present in humans and animals, resulting in lessdilution and higher concentrations of toxic agents. The volume of plasma(5 liters in the adult human male) is much larger than the volume ofsaliva (0.8-1.0 liter produced per 24 hour period), and the larger bloodvolume results in higher dilution of the toxic or reactive agents,making them more difficult to characterize.

Yet another advantage provided by oral saliva biomarkers is that thesecretory cells of salivary glands are innervated by 5-10parasympathetic neurons, and saliva probably contains true enzymes suchas saliva cholinesterase which differs from the pseudo-cholinesterasepresent in plasma. Thus, the inhibition of true acetylcholinesterasefrom saliva is a better indicator for monitoring effects on the nervoussystem by a chemical agent than inhibition of the equivalent enzyme inplasma.

Additionally, collection of saliva does not require invasive proceduresor special equipment as does the collection of tissues, blood, orplasma. The present invention only requires 1-2 mL of saliva forbiomarker enzyme analysis.

Methods for protein fractionation and analysis of human saliva and theuse of human saliva for detecting pregnancy are known. However, thepresence of sensitive biomarkers species such as, but not limited to,acetylcholinesterase, carboxypeptidase, and carboxylase in saliva doesnot appear to have been reported, and the use of enzyme biomarkers fromhuman and animal saliva for characterization of hazardous or toxicagents has not, it appears, heretofore been disclosed.

Accordingly there is a need for a method of detecting, quantifying, andevaluating hazardous agents which provides for early detection, whichcan detect low levels of the agents, and which is not invasive and doesnot require blood or tissue samples from subjects. The present inventionsatisfies these needs, as well as others, and generally overcomes thedeficiencies experienced in the background art.

SUMMARY OF THE INVENTION

The present invention pertains to new and sensitive methods which usesaliva biomarker species from humans and animals to detect, measure, andquantify the presence of infectious and non-infectious hazardous andreactive agents, and to evaluate the functional status of livingtissues. Biomarker species contained in saliva are used in the subjectinvention to evaluate the toxicity of hazardous agents in two broadapplications: (1) in vivo biomedical studies for internal bodilyexposure of people or animals to the agents of interest, and (2) invitro environmental measurements of the presence and/or concentration ofthe various agents of interest in environmental samples such as water,food and the like. The presence of toxic or reactive agents in samplescan be detected, for example, by the degree of inhibition of one or morebiomarker enzymes present in saliva. The saliva provides a source of theenzymes that are inhibited by the agents of interest. For the subjectinvention, the inhibition of biomarker enzymes present in salivaprovides a method of evaluating hazardous agents which appears to bemore sensitive than currently available assay methods.

Generally, the inventive method comprises the steps of acquiring orotherwise providing a saliva sample, analyzing or measuring the levelsor concentrations of one or more biochemical constituents present in thesaliva sample, and comparing the levels of the biochemical constituentsin the sample with baseline levels or concentrations obtained ordetermined from control samples, standards, theoretical quantities, orspecimens. Biomarker species present in the saliva sample, such asenzymes, antibodies, and other proteins, are responsive to various toxicor hazardous infectious and non-infectious agents, and the measuredlevels of biochemical constituents indicate the level of response of thebiomarkers to the hazardous agents and thus the quantity of hazardousagent present.

In a first or in vivo embodiment of the invention, saliva samples aretaken from human and/or animal subjects and biomarkers such as enzymesor antibodies in the saliva are monitored via inhibition or binding.Specifically, the presence of and extent of exposure to hazardous agentsis measured by the presence and amount or level of specific chemical orbiological constituents, by the degree of enzymes inhibition, by theamount of specific binding, or by the changes in the amount of thebiochemical constituents in saliva. Using this embodiment, hazardousagents that inhibit carboxylase, acetylcholinesterase, alkalinephosphatase, acid phosphatase, amylase and other biomarker enzymespresent in oral saliva are detected and quantified. Hazardous agentswhich inhibit these enzymes include, among others, organophosphate andcarbamate pesticides and fluorophosphate nerve agents. The amount ofhazardous agent present in the saliva is determined by comparison to astandard curve for the hazardous agent which is determined from controlsamples or theoretical predictions.

In a second or in vitro embodiment, enzymes or other biomarkers fromanimal or human saliva are used as a ready source for the biomarkers tomonitor the presence of toxic or reactive agent in external orenvironmental samples from tissue, urine, feces, milk, air, water, soil,plants, or other sources. The amount of hazardous agent present insamples is determined from a standard or theoretical inhibition orbinding curve for that agent. In one application of this embodiment,acetylcholinesterase and carboxylase (generally defined as enzymeshaving esterase-like activity) enzymes in human saliva are used todetect the presence of organophosphates such as pesticides in watersamples with exceptional sensitivity.

By way of example and not of limitation, the analyzing step preferablyis carried out by detecting and monitoring the level or concentration ofone or more biochemical constituents in a saliva sample byspectrophotometric or chemical means. The biochemical constituentsanalyzed are indicative of the response of biomarker enzymes to varioushazardous agents. The biochemical constituents may be reactivesubstrates which are specific for the biomarker enzymes, the reactionproducts of the biomarker enzymes and their specific substrates, thebiomarker enzymes themselves, or other chemical or biologicalconstituents present in a saliva sample which indicate the presence andquantity of a hazardous chemical agent. In the presently preferredembodiments, the analyzing step generally involves detecting inhibitionof specific biomarker enzymes by spectrophotometrically monitoring (ormonitoring by an equivalent method) reaction product formation and/ordepletion of reactive substrates within the samples. The analyzing stepalso preferably includes measuring the overall amount of protein (thisoverall amount includes both enzymes and non-enzyme protein components)present in the saliva sample.

The invention may also include the step of determining baseline levelsof biochemical constituents from standard or control saliva samples.Generally, standard curves for biochemical constituents which reflectbiomarker enzyme activity are prepared from control samples. Thedetermining step also preferably includes determining baseline levelsfor the overall protein content of saliva samples.

The in vitro embodiment of the invention preferably also includes thesteps of obtaining or extracting and concentrating hazardous agents fromenvironmental, tissue, or other samples. Purification and concentrationof the saliva sample may also be carried out in this embodiment.Evaluation of the toxicity of new or unknown infectious ornon-infectious hazardous agents may be carried out using the in vitroembodiment.

The present invention has scientific value and has several applicationswith significant commercial potential. The scientific value isexemplified by the discovery of the presence of biomarkers such asacetylcholinesterase and carboxylase enzymes in human saliva and the useof such saliva enzymes to evaluate exposure of human and animals tohazardous agents in both in vivo and in vitro settings. These biomarkerscan also be used to monitor changes in human and animal health thatresult from exposure to endogenous compounds. Purified human and animalsaliva enzymes to be used in clinical, research, industrial, andmilitary applications, as well as by the general public to evaluate the,presence, quantity, and toxicity of agents may be provided.

Test kits can be fabricated that utilize the subject methods withrelated equipment for biomarker enzyme assays. The kits can also be usedto detect residue of chemical and biological toxicants in milk and otherbiological fluids, animal and plant tissues, water, air, and soilsamples. Test kits and equipment may be used by agriculture workers,industrial hygienists, military personnel, researchers, and members ofthe general public.

An object of the invention is to provide a method for detection,quantification, and evaluation of hazardous agents which utilizesbiomarkers present in oral saliva of humans and animals.

Another object of the invention is to provide a method for detection,quantification, and evaluation of hazardous agents which is quick andfacile to use and which does not require expensive equipment orextensive training.

Another object of the invention is to provide a method for detection,quantification, and evaluation of hazardous agents which allowssensitive and accurate characterization of agents present in low levels.

Another object of the invention is to provide a method for detection,quantification, and evaluation of hazardous agents which allows both invivo and in vitro characterization of agents.

Another object of the invention is to provide a method for detection,quantification, and evaluation of hazardous agents which is non-invasiveand does not require blood or tissue samples.

Another object of the invention is to provide a method for detection,quantification, and evaluation of hazardous agents which may be used inkit forms.

Further objects of the invention will be brought out in the followingportions of the specification, wherein the detailed description is forthe purpose of fully disclosing the invention without placing limitsthereon.

BRIEF DESCRIPTION OF THE DRAWINGS

The invention will be more fully understood by reference to thefollowing drawings which are for illustrative purposes only:

FIG. 1 is a flow diagram showing generally the method steps in a firstor in vivo embodiment of a method for using saliva biomarkers to monitorand evaluate toxic agents.

FIG. 2 is a flow diagram showing generally the method steps in a secondor in vitro embodiment of a method for using saliva biomarkers tomonitor and evaluate toxic agents.

FIG. 3 is a graphic representation of a bovine serum albumin absorbancecurve.

FIG. 4 is a graphic representation of a p-nitrophenol absorbance curve.

FIG. 5 is a graphic representation of the activity of carboxylase inhuman saliva versus protein in saliva (μg) in a 4 mL sample.

FIG. 6 is a graphic representation of the activity of carboxylase inhuman saliva versus saliva (μl) in a 4 mL sample.

FIG. 7 is a graphic representation of the percent of carboxylaseinhibition in human saliva by organophosphate pesticide in vitro.

FIG. 8 is a graphic representation of the percent of carboxylaseinhibition in human saliva by organophosphate pesticide.

FIG. 9 is a graphic representation of a glutathione absorbance curve.

FIG. 10 is a graphic representation of the activity ofacetylcholinesterase in human saliva versus incubation time minutes forthree saliva concentrations.

FIG. 11 is a graphic representation of the activity ofacetylcholinesterase in human saliva versus saliva (μl) per 4 mL(incubation time=165 minutes).

FIG. 12 is a graphic representation of the activity ofacetylcholinesterase in human saliva versus protein content in saliva(μg) per 4 mL (incubation time=165 minutes).

FIG. 13 is a graphic representation of the activity ofacetylcholinesterase in human saliva versus volume saliva (mL) in 4 mL.

FIG. 14 is a graphic representation of the activity ofacetylcholinesterase in human saliva versus protein content in saliva(mg) in 4 mL.

FIG. 15 is a graphic representation of the percent ofacetylcholinesterase inhibition in human saliva by DNIO.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT

Referring to FIGS. 1-15, as well as the following detailed description,the present invention is embodied in the process generally describedherein. It will be appreciated that the invention may vary as to detailsand as to sequence in the steps of the process without departing fromthe basic concepts as disclosed herein.

In using the method comprising the present invention, the presence inhuman or animal subjects of and extent of exposure to hazardous agentsis evaluated by analyzing the presence and amount, level, orconcentration of specific chemical or biological constituents(hereinafter collectively referred to as biochemical constituents), bythe degree of inhibition of one or more enzymes, or by the changes inthe amount of the biochemical constituents in saliva. In the presentlypreferred embodiments, the degree of biomarker enzyme inhibition isgenerally followed by detecting and monitoring levels or concentrationsof reaction direct or indirect (or linked) products from particularbiomarker enzymes and their specific substrates. It is noted thatsubstrate usage is acceptable for following the inhibition kinetics ofthe appropriate reaction. The detected levels or concentrations ofreaction products are then compared with baseline levels orconcentrations of the same reaction products in the form of a standardcurve obtained from control or standard samples or specimens.Differences between the monitored and baseline levels of the reactionproducts reflect the degree of inhibition of the biomarker enzymes andthus the presence and amount of the inhibiting hazardous agent.

Referring first to FIG. 1, there is shown a flow chart which generallyoutlines the steps of a first or in vivo embodiment of a method forevaluating, monitoring, and quantifying hazardous agents which may bepresent in a human or animal subject. At step 10, an oral saliva testsample is acquired, provided, or otherwise obtained from a human oranimal subject. The method of obtaining the sample can be varied. Onlyabout one to two milliliters of saliva per test sample are required foruse with the present invention, making this method particularly easy andnon-invasive to carry out. The saliva sample contains numerous naturallypresent biomarkers, in particular for the subject invention, enzymes,antibodies, and other biochemical constituents. The presently preferredbiomarkers employed in the invention include carboxylase,acetylcholinesterase, carboxypeptidase, alkaline phosphatase, acidphosphatase, amylase, and other esterases naturally present in human andanimal saliva.

At step 20, biochemical constituents in the saliva sample are analyzed.Generally, the analyzing step comprises the detection and monitoring oflevels or concentrations of one or more biochemical constituents. Thebiochemical constituents analyzed in this step may include reactivesubstrates specific for biomarker enzymes, the biomarker enzymesthemselves, reaction products of the substrates and biomarker enzymes,or any proteins or biomolecules present in the saliva sample. Thebiochemical constituents analyzed may be naturally present in the salivasample or added thereto during the analyzing step, as in the case ofcertain easily detectable substrates and substrate reaction products.Preferably, the biochemical constituents analyzed in this step are oneswhich may be detected and monitored by spectrophotometric orcalorimetric means or other easily applicable techniques that arereadily usable in laboratory and non-laboratory or field conditionswhere sophisticated instruments are limited. Standard or later developedchemical detection and monitoring means may also be employed. Thedetection is preferably by visible spectrophotometry, allowingmonitoring of sample optical density or transmission at wavelengths forspecific chemical or biochemical constituents.

As related above, the presently preferred in vivo embodiment involvesmonitoring the inhibition of biomarker enzymes by hazardous agents bydetection and monitoring reaction products of the biomarker enzymes withspecific substrates. For reasons of clarity, the analyzing step is shownas sub-steps 22, 24, and 26 are shown in the analyzing step 20 to moreclearly show the presently preferred in vivo embodiment. As used in thisdisclosure, the term in vivo implies the monitoring of inhibition ofsalival biomarker enzymes by hazardous agents that are directly presentin the sample saliva or elsewhere in the body as opposed to in vitrocases where the utilized saliva has the hazardous agent or agentsintroduced after acquisition for measurement purposes.

At sub-step 22, the overall protein content of the saliva sample isanalyzed and measured. The protein content measurement is generallycarried out on an aliquot of the saliva sample by standard visiblespectrophotometric means as related by Lowry et al. in “ProteinMeasurement with the Folin Phenol Reagent”, J. Biol. Chem. Vol. 193, pp.265-275 (1951), which is herein incorporated by reference. It is notedthat other equivalent protein determination methods are acceptable.Generally, bovine serum albumin or gamma-globulin are used as referencestandards, with the protein concentration values determined bycomparison with a standard curve. Human saliva generally contains about0.2-0.5% protein by weight. This protein measurement is necessary forcalculating the specific activity of the biomarker enzymes monitored inanalyzing step 20. The specific activity of the biomarker enzymes is arelative measurement based on the total protein concentration and notnecessarily the concentration of any one specific enzyme of enzymes.

At sub-step 24, one or more reactive substrates specific for one or morebiomarker enzymes are added to the saliva sample, to serve asbiochemical constituents to be analyzed directly or via linked assayprocedures common in the art. Preferably, the added reaction substratemeasure directly and is therefore one which forms easily detectablereaction products upon reaction with its specific biomarker enzyme. Fora linked assay, additional chemicals or indicators may be added in thisstep to form biochemical constituents which are easily detected. In oneapplication of the in vivo embodiment, as related in the experimentalsection below, the substrate p-nitrophenylvalerate, which is specificfor the biomarker enzyme carboxylase, is added to the saliva sample andforms the hydrolysis product p-nitrophenol which can be detected andmonitored spectrophotometrically at 400 nm. In another application ofthis embodiment, the substrate acetylthiocholine iodide is added to thesaliva sample, and is hydrolyzed by acetylcholinesterase to yield, afterthe addition of 5,5-dithiobis-2-nitrobenzene, a colored species whichcan be detected spectrophotometrically at 405 nm. It is alsocontemplated that substrates and their reaction products which arenaturally present in saliva could be detected and monitored.

The analyzing step 20 generally includes an incubation sub-step 26,wherein the saliva sample and the added reaction substrates or otheradded biochemical constituents are allowed to react prior to detectionand monitoring of reaction products. Preferably, though not critically,samples are incubated at about 37° C. in a suitable temperaturecontrolled bath. The incubation period and temperature may varydepending upon the reactive substrate and biomarker enzymes beinganalyzed. Ambient temperature incubation or no incubation at all maysuffice, again depending upon the nature of the biochemical constituentsto be analyzed.

At sub-step 28, the reaction products from the specific substrate andbiomarker enzyme are detected and monitored, usually byspectrophotometric means, preferably by visible spectrophotometric meansas related above. The detection and monitoring indicates the level orconcentration of the reaction products or other biochemical constituentspresent in the saliva sample (or in the case of reaction substrateevaluations the level or concentration of the substrate). The activityof the biomarker enzyme in the saliva sample can be calculated from thelevel or concentration of reaction product (or substrate) obtained inthis sub-step.

At step 30, the levels or concentrations of the biochemical constituentsare compared to standard or baseline levels for the biochemicalconstituents which are obtained from control or standard samples orspecimens. The standard or base line level is preferably in the form ofa standard curve showing the level, concentration, or othercharacteristic of the biochemical constituents analyzed in the previousstep 20. In the preferred embodiment, the standard curve is one forbiomarker enzyme activity which is obtained by inhibition studies in aplurality of control saliva samples. Comparison of the biomarker enzymeactivity from the test sample to the standard curves accuratelyindicates the presence and amount of particular hazardous agents.

The in vivo embodiment may also include the step of determining the baseline levels of the biochemical constituents (not shown) to be used inthe comparing step 30. This step is generally carried out by adding testcompounds or hazardous agents to control samples and incubating,followed by analyzing biochemical constituents in the sample, preferablyin the form of substrate reaction products as related above. A biomarkerenzyme activity standard curve is prepared from data from the controlsamples, and used in the comparing step 30.

Using the in vivo embodiment of the invention above, sensitivebiomarkers can be used to monitor the exposure of people and animals toenzyme inhibiting toxic or reactive agents at low levels and it can beused to quantify the extent of exposure at a stage when the process ishopefully readily reversible. In particular, human saliva containsmeasurable activity of acetylcholinesterase and carboxylase biomarkerenzymes. The activities for carboxylase and acetylcholinesterase areabout 47-79 and about 4 nmol min⁻¹ per mL of saliva, respectively, asrelated below in the experimental section. These two biomarker enzymesare inhibited by isofenphos (IFP), des N-Isofenphos-oxon (DNIO) andparaoxon organophosphates. The effective concentration for 50%inhibition EC₅₀ for both carboxylase and acetylcholinesterase by DNIO is3 ng/mL. The detection limit of DNIO by gas chromatography is 10,000ng/mL, in comparison. Detection of DNIO using saliva biomarker enzymesis thus 3500 time more sensitive than measuring DNIO by gaschromatography (GC). These data indicate that the enzymes in saliva ofhuman and animals can be used with the present invention as verysensitive biomarkers to evaluate the degree of exposure to toxicants andto quantify the extent of exposure at a stage when the process isreadily reversible.

There are numerous other hazardous agents which inhibit carboxylase andacetylcholinesterase and which may be detected, quantified, andevaluated in vivo using the present invention. For example, human oranimal exposure to other organophosphate insecticides, carbamateinsecticides, blue green algae-anatoxicant-A, solanum specious blacknightshade (S. nigrum), potato (S. tuberosum), horse nettle (S.tuberosum), European bittersweet (S. dulcamara), Jerusalem cherry (S.pseudocapsicum), as well as other noninfectious and infectious hazardousagents.

While the preferred in vivo embodiment of the invention as related aboveis described generally as it is used to detect and quantify inhibitionof carboxylase and acetylcholinesterase by organophosphates, it shouldbe readily apparent to persons skilled in the art that the invention maybe used in evaluating a variety of infectious and non-infectioushazardous agents. Examples of hazardous noninfectious agentscontemplated for detection, quantification, or evaluation with thepresent invention include carbamates and other pesticides, nerve agents,mustard agents, biotoxicants, heavy metals, and endogenous compounds.Hazardous infectious agents considered for evaluation with the presentinvention include viruses, mycoplasmas, bacteria, parasites, yeast, andfungus. These hazardous agents have detectable effects on variousbiomarkers present in saliva, and may be evaluated using the in vivoembodiment.

Biomarkers other than enzymes may also be employed with the invention.For example, antibodies present in saliva may be analyzed for bindingwith antigens to evaluate the toxicity in humans and animals ofinfectious agents. The in vivo embodiment of the invention may also beused to evaluate toxicity in humans and animals from endogenouscompounds resulting from organ malfunctions, such as acholinesterasemiain the liver. The presence of various biochemical constituents in salivacan be used to evaluate various tissue and organ malfunctions orpotential malfunctions. The in vivo embodiment of the present inventionthus provides a powerful medical diagnostic method which is easy to useand is non-invasive, requiring only small amounts of saliva samples fromsubjects.

Referring next to FIG. 2, there is shown a flow diagram which generallyoutlines the steps of a second or in vitro embodiment of the presentinvention. In this embodiment, biomarkers in saliva samples are used toevaluate hazardous agents present in external or in vitro samples fromsources other than the saliva containing the biomarker enzymes, ratherthan hazardous agents present in vivo in the saliva or in the body ofhuman and animal subjects. As with the in vivo embodiment related above,the degree of biomarker enzyme inhibition is preferably monitored bydetecting and monitoring levels or concentrations of reaction products(or substrate concentration) from particular biomarker enzymes and theirspecific substrates. The detected levels or concentrations of reactionproducts are then compared with baseline levels or concentrations of thesame products in the form of a standard curve obtained from control orstandard samples or specimens. Differences between the monitored andbaseline levels of the reaction products reflect the degree ofinhibition of the biomarker enzymes and thus the presence and amount ofthe inhibiting hazardous agent.

At step 100, a saliva sample is acquired, obtained, or otherwiseprovided by any suitable technique. As in the in vivo embodiment, onlyabout one to two milliliters of saliva per test sample are usuallyrequired. The same naturally present biomarkers related above may beemployed in the in vitro embodiment. As above, the activity orinhibition of activity of a collection of similarly acting enzymes(esterase or esterase-like functions) may be utilized in the analysis.

At step 110, biochemical constituents in the saliva sample arecharacterized. This step generally includes measuring the overallprotein content of the sample as related above in the in vivoembodiment, preferably using the technique outlined by Lowry et al or inthe alternative any suitable process. Since the saliva samples in the invitro embodiment are used to characterize hazardous agents from externalor outside sources (not directly in the acquired saliva sample), thebiomarkers or other biochemical constituents to be analyzed, detected,and monitored in this embodiment are generally characterized to insurethat the saliva sample is free from hazardous agents which may interferewith subsequent analysis. For example, the relative specific activity(relative to a selected protein concentration which may or may not beonly the catalyzing enzyme concentration) of particular biomarkerenzymes may be determined in this step if the particular enzymes are tobe analyzed or monitored in subsequent steps.

Step 110 may include an additional purification step (not shown) whereinthe saliva sample is purified to remove water or certain chemical orbiochemical constituents which may interfere with subsequent steps.

At step 120 a hazardous agent-containing sample, or a sample whichpotentially contains hazardous agents, is acquired, obtained, orotherwise provided. The hazardous agent-containing specimens or samplesmay be obtained from milk, urine, feces, blood, plasma, body tissue,water, food, air, soil, another saliva sample, or other source. Step 120may also comprise extraction, separation, concentration, or purificationof the hazardous-agent-containing sample. Such extraction andconcentration can take many forms. For example, air samples whichpotentially contain airborne hazardous agents may be bubbled throughwater or other aqueous or non-aqueous solution, followed by passing thewater through a solid phase absorption column with a hydrophobic phasesuch as a cyclohexyl solid phase, and eluted through with alcohol orother organic solvent. Hazardous agent-containing samples may similarlybe obtained from water, soil, biological fluids, and tissues byextracting with organic or aqueous solvents and absorption columnseparation, followed by solvent removal. A method for extracting samplesfrom tissue specimens is related by Knaak et al. in “Development of inVitro V_(max) and K_(m) Values for the Metabolism of Isophenos by P-450Liver Enzymes in Animals and Human”, Toxicology and AppliedPharmacology, Vol. 120, pp. 106-113 (1993), which is herein incorporatedby reference.

At step 130, the hazardous (or even non-hazardous if desired)agent-containing sample is combined with or added to the saliva sample,and the combined samples are incubated. The incubation in this stepallows hazardous agents to interact with biomarkers. The incubationpreferably is carried out at about 37° C. in a water bath, but otherappropriate temperatures are contemplated to within the realm of thisdisclosure. As with the in vivo embodiment, the incubation time may bevaried depending upon the particular biomarkers and chemical orbiochemical constituents to be analyzed.

At step 140, chemical or biochemical constituents present in thecombined samples are analyzed. The analyzing step generally comprisesthe detection and monitoring of levels or concentrations of one or morebiochemical constituents. As in the in vivo embodiment related above,biochemical constituents analyzed in this step may include reactivesubstrates specific for biomarker enzymes, the biomarker enzymesthemselves, reaction products of the substrates, substrates themselves,or any chemicals, proteins, or biomolecules present in the salivasample. Preferably, spectrophotometric or chemical detection andmonitoring means are employed.

The in vitro embodiment, as with in vivo embodiment, preferably involvesmonitoring the inhibition of biomarker enzymes by hazardous agents bydetection and monitoring the reaction product(s) or substrate(s) of thebiomarker enzymes with a specific selected substrate or substrate.Again, for reasons of clarity, analyzing step 140 is shown as sub-stepsto illustrate the presently preferred in vitro embodiment.

At subs-step 142, one or more reactive substrates specific for or atleast acted upon by one or more biomarker enzymes are added to thecombined samples from step 130, to serve as biochemical constituents tobe analyzed. The added reaction substrate preferably forms easilydetectable reaction products upon reaction with its specific biomarkerenzyme. One preferred use of the in vitro embodiment involves additionof the substrate p-nitrophenylvalerate, which reacts with the biomarkerenzyme carboxylase and forms the hydrolysis product p-nitrophenol whichcan be detected and monitored spectrophotometrically at 400 nm.Similarly, the substrate acetylthiocholine iodide may be added to thecombined samples and hydrolyzed by acetylcholinesterase to yield, uponreaction with added 5,5-dithiobis-2-nitrobenzene, a product detectableat 405 nm.

An incubation sub-step 144 is preferably included in analyzing step 140.The combined samples, together with the added substrates, are incubatedat about 37° C. (or other suitable temperature) in a temperaturecontrolled bath for an incubation period which is varied depending uponthe reactive substrate and biomarker enzymes being analyzed. Further,ambient temperature incubation or no incubation at all may suffice.

At sub-step 146, the reaction products from the specific substrate andbiomarker enzyme are detected and monitored, usually by visiblespectrophotometric means as related above. The detection and monitoringindicates the level or concentration of the reaction products, reactionsubstrates, or other biochemical constituents present in the salivasample. The activity of the biomarker enzyme in the combined samplesfrom step 130 can be calculated from the level or concentration ofreaction product or remaining substrate obtained in this sub-step.

At step 150, the levels or concentrations of the biochemicalconstituents are compared to standard or baseline levels for thebiochemical constituents which are obtained from control or standardsamples or specimens. The standard or base line level is preferably inthe form of a standard curve. In the preferred in vitro embodiment, thestandard curve is one for biomarker enzyme activity which is obtained byinhibition studies in a plurality of control saliva samples. Comparisonof the biomarker enzyme activity from the test sample to the standardcurves accurately indicates the presence and amount of particularhazardous agents.

The in vitro embodiment may also include the step of determining thebase line levels of the biochemical constituents to be used in thecomparing step 30. As in the in vivo embodiment, this step is generallycarried out by adding test compounds or hazardous agents to controlsamples and incubating, followed by analyzing biochemical constituentsin the sample, preferably in the form of substrate reaction products asrelated above. A biomarker enzyme activity standard curve is preparedfrom data from the control samples, and used in the comparing step 150.

Using the in vitro embodiment of the present invention, hazardous agentspresent from in vitro or otherwise external sources can be detected,quantified, and evaluated by adding the hazardous agent-containingsamples to previously characterized saliva samples. Preferably, thebiomarkers employed in the in vitro embodiment are acetylcholinesteraseand carboxylase (or carboxypeptidase or a combination of these and otherenzymes), which are inhibited by IFP, DNIO, and paraoxonorganophosphates. The in vitro embodiment allows very sensitivedetection of low amounts of these materials in air, water, soil,biological fluid samples, and similar materials. The several otherhazardous agents related above which inhibit acetylcholinesterase andcarboxylase may also be detected and quantified using the in vitroembodiment.

While the in vitro embodiment is described herein for the inhibitionacetylcholinesterase and carboxylase (and usually other similarly actingenzymes present in the saliva), it is contemplated that the in vitroembodiment, like the in vivo embodiment, may also be used to evaluate avariety of hazardous agents through analysis of a variety of biochemicalconstituents. Thus, the in vitro embodiment of the invention may beemployed to detect a variety of hazardous agents, includingorganophosphates, carbamates, or other pesticides, nerve agents, mustardagents, biotoxicants, heavy metals, endogenous compounds, viruses,mycoplasmas, bacteria, parasites, yeast, and fungus. These agents may bedetected in environmental specimens such as air, water, and soil, infood samples, and in biological fluid and tissue samples. The in vitroembodiment can be used to detect and evaluate organ or tissuemalfunctions by extracting samples from tissue or biological fluids andcombining with a purified and characterized saliva sample as relatedabove, and detecting endogenous compounds resulting from themalfunction. Residual hazardous agents in tissue or biological fluids,and toxic metabolites of the hazardous agents may also be evaluatedusing this embodiment.

A kit may be provided for home or field use of the in vitro embodimentof the invention. Such a kit would include, in the case of carboxylase(or other like enzyme or combination of enzymes) as a biomarker forexample, suitable p-nitrophenylvalerate or equivalent solution, suitablebuffer solutions, p-nitrophenol standard solution, and purifiedcarboxylase (or other like enzyme or combination of enzymes) fromsaliva. A portable constant-temperature bath and visiblespectrophotometer may also be included, but the kit may be utilizedwithout a constant-temperature bath.

EXPERIMENTAL

I. Measuring Carboxylase (CE) Activity in Saliva and EC₅₀ for Inhibitionby Pesticides

The protein in 50 μL aliquot of saliva is measured by using bovine serumalbumin as a reference standard (Lowery et al., 1951). Briefly, a colorwas developed by the reaction of protein in saliva and the reagents. Thesample was read by a spectrophotometer at 540 nm. The values for theprotein standard curve and protein in saliva are shown in Tables 1-2 andFIG. 3. Saliva generally contains about 0.2-0.5% protein, and the exactprotein measurement (content) in saliva is needed for calculating thespecific activity for the enzymes (specific in the sense of specific tothe total measured protein concentration or volume of saliva used).

The carboxylase activity in saliva samples is monitored by thehydrolysis of p-nitrophenylvalerate to the readily detectablep-nitrophenol. The concentration of p-nitrophenol in the sample ismeasured spectrophotometrically at 400 nm, and the value is calculatedfrom p-nitrophenol standard curve (Table 3 and FIG. 4). Values forcarboxylase activity in saliva (nmol. min-1 per mL of saliva) arepresented in Tables 4-8 and showing in FIG. 5 and FIG. 6. The averagespecific activity for carboxylase in human saliva is about 47 nmol min⁻¹per mL.

The assay for carboxylase consists of 4 mL of tris-HCl buffer (pH 7.0)that contain 25 μL of p-nitrophenylvalerate (13.8 mg/mL of alcohol) and50-500 μL of human saliva. The sample is incubated in water bath forabout 20-60 min at about 37° C. In the inhibition study, the agent isincubated with saliva for about 15-20 minutes before the addition ofp-nitrophenylvalerate. The values for percents enzyme inhibition byorganophosphate are presented in Tables 5-8 and shown in FIG. 7 and FIG.8. Organophosphate concentration is verified by gas chromatography usingstandard methods as described by Knaak et al. (1993). DNIO concentrationproduced 50% inhibition is 2.9 ng per mL (3 PPB).

The use of carboxylase in human saliva to detect the presence of DNIO inwater or biological fluid (3 ng/ml) is 3500 times more sensitive thanmeasuring DNIO by GC method.

The kit for carboxylase assay in saliva using this invention willusually consist of p-nitrophenylvalerate or an equivalent as asubstrate, tris-HCl buffer (pH 7.0) or an equivalent, purifiedcarboxylase enzyme from human or animal saliva (1 Unit),andp-nitrophenol as standard. The test is performed at about 37° C. in awater bath or at room or other suitable temperature. By way of example,each test may requires 0.35 mg of p-nitrophenylvalerate in 3 mL ofphosphate buffer, pH 7.0 and about 0.2-1 mL of saliva. Sample can beincubated at room temperature, about 37° C., other selected temperaturein a water bath for about 10-30 minutes. Generally, samples are readusing a spectrophotometer at 400 nm. For quality control, 0.05 IU (orother acceptable amount) of purified human or animal saliva enzyme isused as a standard per assay. For monitoring the presence of pesticidesin milk, biological fluids, water, and other environmental samples, thecyclohexyl solid phase absorption column (or like procedure) is used toextract and concentrate the chemicals utilizing standard methods asdescribed by Knaak et al. (1993). P450 liver enzymes and cofactors areused to convert organic phosphothionate to oxon as described by Knaak etal. (1993) to increase sensitivity of the procedure and to decrease thedetection limit for this class of chemicals and other chemicals thatrequires activation by liver enzymes.

TABLE 1 Bovine Serum Albumin (BSA) Absorbance data Sample # BSA μg/tube(A) at 540 nm (B) at 540 nm Average 1 25 0.158 0.158 0.158 2 50 0.2820.285 0.284 3 75 0.420 0.412 0.416 4 100 0.520 0.530 0.525 5 125 0.6400.630 0.635

TABLE 2 Protein Content of Human Saliva* μg Sample (A) at (B) at (C) atprotein/ mg protein/ # 540 nm 540 nm 540 nm Mean sample mL % IC1 .343.435 .343 .374 71.2 2.848 .28 IC2 .62 .702 .795 .706 134.5 5.379 .54 IC3.502 .540 .560 .534 101.7 4.069 .41 IC4 .415 .450 .420 .428 81.5 3.261.33 IC5 .202 .220 .210 .211 40.2 1.608 .16 *Saliva samples were obtainedfrom a 41 years old, adult male. A volume of 25 μL of each sample wasused for each assay.

TABLE 3 p-nitrophenol Absorbance Data Sample # μg/tube nmol/tube (A) at400 nm (B) at 400 nm Average Blank 0 1 1.7 7.19 .04 .036 .038 2 3.514.38 .083 .088 .086 3 7.0 28.76 .160 .158 .159 4 14 57.51 .330 .330.330 5 21 86.27 .518 .518 .518 6 28 115.03 .750 .755 .753 *p-nitrophenol0.125 mL (10 μmol/mL, molecular weight = 139.1) in 25 mL 0.02 N NaOH.,Sigma lot # 110H5005, C₆H₅NO₃. Final concentration = (0.125* 10)/25 = 50nmol/mL, or 6.95 μg/mL.

TABLE 4 Carboxylase Activity In Human Saliva* (A) at (B) at Net μL μgActivity Tube # 400 nm 400 nm Average saliva** protein** nmol/min 1 .01.01 0 0 0 0 2*** .03 .02 0 200 570 0 3 .08 .09 .06 20 57 .44 4 .17 .14.13 40 114 .96 5 .22 .28 .22 60 171 1.70 6 .42 .37 .38 80 228 2.89 7 .63.60 .59 100 285 4.48 8 1.00  .94 .94 150 427 6.99**** 9 2+ 2+ 2+ 200 57015.28 *Incubation time 20 minutes and volume of media was 4 mL inphosphate buffer, pH 7.0. **Protein concentration in saliva = 2.848mg/mL ***No substrate ****Carboxylase Activity (nmol. min⁻¹) per mgprotein = 18.8

TABLE 5 Inhibition of Carboxylase in Human Saliva by pesticides (test#1)* Absorb. at Absorb. at 400 nm 400 nm Sample Organophos. for for % #Treatment μg/ml 20 min. 60 min. Inhib. 10 Paraoxon in 29 .03 .14 99Tris/Citrate 11 Tris/Citrate 0 .58 2 71 12 Alcohol 0 2**  2 0 13Isofenphos 500 .33 .75 84 14 DSN- 175 .16 .55 92 Isofenphos- oxon *A 200μL of saliva (2.484 mg protein/mL) was incubated with the test compoundfor 15 minutes, then 25 μL of p-nitrophenylvalerate (13.8 mg/mL) wasadded to the mixture, and the mixture was incubated for 20-60 minutes at37° C. The organophosphate samples was analyzed by gas chromatography.**Carboxylase activity (nmol minute⁻¹ per mg protein) = 31.0

TABLE 6 Inhibition of Carboxylase in Human Saliva by Pesticides (Test#2)* Absorb. Absorb. at 400 at 400 Absorb. at Organo- Sample nm for nmfor 400 nm for phosphate % # 20 min. 65 min. 100 min. treatment μg/mLAct. 1 0 0 0 Blank 0 100 3** .53 +2.0 2.0 Alcohol 0 4 .60 2.0 2.0Isofenphos 60 92 5 .47 1.1 1.5 Isofenphos 60 6 .30 .55 .64 DNIO 5 35 7.27 .57 .69 DNIO 5 8 .23 .68 .76 Paraoxon 22 40 9 .23 .70 .76 Paraoxon22 *A 50 μL of saliva (4.069 mg/mL) was used in 4 mL phosphate buffer pH7.0 with a 25 μL of p-nitrophenylvalerate (13.8 mg/mL). Saliva samplewas incubated with organophosphate or alcohol for 20 minutes before theaddition of substrate. **Carboxylase activity (nmol minute⁻¹ per mgprotein) = 17.0

TABLE 7 Inhibition of Carboxylase ln Human Saliva byDESN-Isofenphos-Oxon (Test #1)* Absorb. at Absorb. at Absorb. at 400 nmfor 400 nm for 400 nm for Sample # DINO ng/mL 20 min. 40 min. 80 min %Activity % Inhibition 1 Blank 0 0 0 0 2 Blank 0 .01 0 0 3 0 .17 .33.62** 100 0 4 625 .05 .07 .10 16 84 5 312 .04 .06 .08 13 87 6 156 .02.02 .03 5 95 7 78 .02 .02 .02 3 97 8 52 .03 .03 .03 5 95 9 26 .02 .03.02 3 97 10 0 .2 .20 .65 100 0 *A 100 μL of saliva (1.608 mg protein/mL)was added to the mixture. The saliva was incubated with DNIO for 15minutes at 37° C. before adding 25 μL p-nitrophenylvalerate (13.8 mg/mLof alcohol). Total volume of media was 4 mL in phosphate buffer pH 7.0**Carboxylase activity (nmol minute⁻¹ per mg protein) = 7.4

TABLE 8 Inhibition of Carboxylase In Human Saliva by DSN-Isofenphos-Oxon(Test #2)* nmol/min Sample DINO Absorb. at per mg % % # ng/ml 400 nmprotein Activity Inhibition 1 Blank 0 0 0 2 Blank 0 0 0 3 0 .8 13.09 1000 4 0 .8 13.09 100 0 5 8.6 .32 5.24 40 60 6 2.9 .40 6.55 50 50 7 1.0 .467.46 57 43 8 .3 .62 10.21 78 22 9 .1 .66 10.87 83 17 10 .04 .75 12.31 946 *A 0.2 mL of saliva (1.608 mg/mL) was incubated for 15 minutes withDNIO at 37° C., then 25 μL of p-nitrophenylvalerate (13.8) was added tothe mixture and incubated for 30 minutes. Volume of media = 4 mLphosphate buffer pH 7.0

II. Procedure for Measuring Acetylcholinesterase (AChE) Activity inSaliva and EC₅₀ for Inhibition by Pesticides

The hydrolysis of acetylthiocholine iodide (an analog of acetylcholineiodides) by the saliva enzyme or enzymes is measured by generating theyellow 5-thio-2-nitrobenzoate with an absorbance maximum at 405 nm. Therate of change of absorbance is directly proportional toacetylcholinesterase activity. In this procedure, 0.1-1 mL of saliva(3.261 mg protein/mL) is incubated with 100 μL of acetylthiocholineiodide (0.018M) (lot #10H0653 Sigma) for 30-180 minute at 37° C. in 4.0mL phosphate buffer (pH 7.8). After the incubation, a 0.2 mL of DTNB isadded and the sample is read at 405 nm. The enzyme activity iscalculated from glutathione standard curve (Table 9 and FIG. 9). Valuesfor AChE activity in human saliva and enzyme kinetics are presented inTables 10-14 and show in FIG. 10 through FIG. 14.

The AChE specific activity in saliva is about 4 nmol minute⁻¹ per mL ofsaliva. In the inhibition study for AChE by organophosphate, saliva isincubated with the inhibitor for about 15 minutes before adding thesubstrate. The percent of enzyme inhibition by organophosphate iscalculated and presented in Tables 13-14 and showing in FIG. 15. A 3 ngDNIO per mL produced 50% inhibition in AChE which is 3500 times moresensitive than the delectability limit for DNIO by gas chromatography(10,000 ng/mL).

Monitoring the presence of pesticides in milk, biological fluids, water,and other environmental samples may require the use of cyclohexyl solidphase absorption column to extract and concentrate the chemicals such asdescribed by Knaak et al. (1993). P-450 liver enzymes and cofactors isused to convert organic phosphothionate to oxon such as described byKnaak et al.(1993) to increase sensitivity of the procedure and todecrease the detection limit for this class of chemicals and otherchemicals that requires activation by liver enzymes. The exposure toother agents that inhibit cholinesterase are also evaluated by thisprocedure. These agents include: (a) Other organophosphate insecticides,(b) carbamate insecticides, (c) blue green algae-anatoxicant-A (s), (d)solanum specious black nightshade (S. nigrum), potato (S. tuberosum),horse nettle (S. tuberosum), European bittersweet (S. dulcamara),Jerusalem cherry (S. pseudocapsicum)}, (e) other noninfectious agents,and 6) infectious agents.

TABLE 9 Glutathione (GSH) Standard Curve* Absorb. at Absorb. at Sample #405 nm (A) 405 nm (B) Average GSH μg GSH nmol 1 .072 .070 .071 3.84 12.52 .182 .187 .185 7.68 25.0 3 .188 .240 .214 11.52 37.5 4 .258 .33 .29415.37 50.1 5 .360 .39 .375 23.05 75.1 6 .435 .480 .447 30.73 100 7 .820.820 .820 61.46 200 *Glutathione = 0.0154 g/50 mL EDTA working buffer or15.4 mg/50 mL.

TABLE 10 Carboxylase Activity in Human Saliva sample used forCholinesterase measurement p-nitro- Absorb. at Absorb. at Absorb. atAbsorb. at Phosphate phenyl- 400 nm for 400 nm for 400 nm for 400 nm forSample # Saliva μL* Buffer mL valerate μL 5 min. 15 min. 22 min. 40min.** 1 50 3.95 0 0 0 0 0 2 50 3.95 25 .1 .35 .48 1.0 *Proteinconcentration = 3.261 mg/mL, 0.163 mg per sample. **Carboxylase Activity(nmol minute⁻¹ per mg protein) = 24.1 and Carboxylase Activity (nmolminute⁻¹ per mL of saliva) = 78.6 or 0.8 IU per 100 mL.

TABLE 11 Acetylcholinesterase Activity In Human Saliva (test #1).Absorb. at Sub. DTNB Buffer Saliva 405 nm for Tube # μL μL mL ml* 55min. 1 100 100 3.8 0 0 2 100 100 3.8 0 0 3 100 100 2.8 1 More than 2** 4100 100 2.8 1 1.5 *Protein concentration in saliva = 3.261 mg/mL, samplewas incubated at 37° C. **Acetyl Cholinesterase Activity (nmol. min⁻¹per mg protein) = 2.183

TABLE 12 Acetylcholinesterase Activity in Human Saliva (Test #2) Absorb.at Absorb. at Absorb. at 405 nm for 405 nm for 405 nm for Activity inSample # Saliva μL Treat. Type 50 min. 110 min. 165 min. nmol/min 1Blank 0 .01 .01 0 0 2  25 0 .03 .02 .03 .06 3  50 0 .08 .08 .10 .20 4 75 0 .10 .12 .17 .34 5 100 0 .16 .21 .29 .58 6 125 0 .18 .26 .36 .72 7150 0 .22 .30 .43 .86 8 175 0 .24 .37 .50 1.00 9 200** 0 .27 .42 .571.14 10 200 IFP*** .41 .43 .51 1.02 11 200 DNIO .14 .10 .11 .22*Substrate: Acetylthiocholine iodide, 0.018M (lot #10H0653, Sigma),0.2603 g in 50 mL distilled water = 5.206 mg/mL; 100 μL was used.Protein concentration in saliva = 3.261 mg/mL. Incubation media is 4.0mL of phosphate buffer (pH 7.0), and incubation temperature was 37° C.**Cholinesterase activity (nmol. min⁻¹ per mg protein) = 1.09***Inhibitor volume = 25 μL contain 240 μg of isofenphos or 2 μg ofDNIO.

TABLE 13 Inhibition of Acetylcholinesterase In Human Saliva by DESN-ISOFENPHOS OXON (Test #1) Absorb. at 405 nm Absorb. at Absorb. at SalivaInhibitor (n = 2) 405 nm 405 nm Tube #* mL mL for 35 min. for 55 min.for 100 min. 1 0 DTNB 0 0 0 2 1 DTNB .06 0 0 3 .2 0 .11 .14 .21 4 .3 0.22 .24 .38 5 .4 0 .18 .24 .44 6 .5 0 .23 .33 .55 7 .6 0 .26 .42 .73 8.8 0 .36 .55 1.30 9 1.0 0 .46 .75 2.00 10 1.0 0 .55 .85 2.00 11 1.0DNIO** .04 .08 .03 12 1.0 DNIO   .07 .04 .03 *100 μL ofAcetylthiocholine solution was added to phosphate buffer (pH 7.8), totalvolume = 4 mL. **A 25 μL of DNIO (4 μg) solution was added to mixture.

TABLE 14 Inhibition Acetylcholinesterase In Human Saliva by DESN-Isofenphos-Oxon (Test #2)* Absorb. at Absorb. at 408 nm for 408 nm fornmol/min per Sample # DNIO ng/mL 65 min. 180 min. mg % Activity %Inhibition 1 0 .01 .01 2 0 .19 .32 .41 100 0 3 0 .19 .31 .41 100 0 4 625.07 .03 .04 10 90 5 313 .07 .03 .04 10 90 6 156 .10 .04 .05 13 87 7 78.10 .04 .05 13 87 8 52 .09 .04 .05 13 87 9 26 .11 .06 .08 19 81 10 8.6.11 .08 .11 26 74 11 2.9 .16 .14 .19 45 55 12 1.0 .17 .21 .29 68 32 13.3 .21 .26 .34 84 16 14 .1 .14 .28 .37 90 10 15 .04 .17 .29 .39 94 6*DESN-Isofenphos-Oxon (DNIO) was incubated for 30 minutes with salivabefore the addition of acetylthiocholine substrate at room temperature.Total volume = 4 mL phosphate buffer (pH 7.8), saliva volume = 0.6 mL,protein concentration = 1.608 mg/mL, protein per incubation = 1.008 mg.

III. Monitoring Milk, Biological Fluids, Water, and EnvironmentalSamples for Toxicants Using Saliva Biomarkers.

The presence of chemicals in milk, water, urine and other biologicalfluids are detected using saliva enzymes. The detection limit for thechemicals depends on the chemical potency and the number of chemicalspresent in the sample. The data presented here using saliva enzymesindicates a remarkably low concentration of des N-isofenphos (DNIO) inwater (3 ng/mL) is detectable using the saliva enzyme method. A 50%inhibition of esterase activity is produced at 3 ng/mL DNIO. In thisprocedure, the sample is passed through cyclohexyl solid phaseabsorption column (C-18) to extract and concentrate the chemicals asdescribed by Knaak et al. (1993). The retention of the chemicals on C-18column depends on their lipid solubility. The non-polar compoundsusually have the highest retention. Then, the chemical is eluted with 1mL of alcohol. A 0.1 mL of the elute is tested for the inhibition ofsaliva enzymes as described in this application. Toxicants in solidenvironmental samples, fruit and vegetables can be extracted with wateror proper solvent. Samples of the extract are tested for enzymesinhibition. The inhibition of saliva enzyme indicates the presence oftoxicant(s) in the sample and further analysis is needed to identify thespecific chemical(s).

IV. Using Saliva Biomarkers to Evaluate Toxicity of Chemical andBiological Agents In Vitro.

Purified saliva enzymes from human and animals can be used asinexpensive and sensitive tools to screen for toxicity of agents thatinhibit one or more enzyme system in saliva. This procedure can be usedin research and in chemical production setting replacing the need ofusing experimental animals. The use of animals in toxicity testing isexpensive and time consuming when compared with the use of the salivaenzymes. In addition, the data collected from animal studies may notaccurately predict human toxicity. Using the saliva methods, up to 60samples can be assayed for enzymes inhibition within one hour.

V. Monitoring the Presence of Toxic or Reactive Agents in Air.

Toxic or reactive agents in air can be detected by the degree of theinhibition of enzymes or alteration of other biochemicals in saliva inboth in vivo biomedical applications and in vitro environmentalapplications. In the in vivo applications enzymes or other biochemicalbiomarker activities in saliva are collected from a subject beforeentering the work area and during and after leaving the work area. Inthe in vitro environmental applications air samples are collected in abubbler system containing the suitable trapping solvent, on suitablefilters, or by other suitable means. The presence of chemicals thatinhibit saliva enzymes or react with other saliva biochemicals areassayed using standard instrumental or chemical techniques. For example,enzyme activity in the presence of pure trapping solvent can assayed andused as a reference standard.

VI. Monitoring the Functioning of Organs

Additionally, the subject invention has use as a method for evaluatingorgan function by monitoring changes in the chemical composition ofsaliva. By following the presence and concentration (increases ordecreases) of various components in saliva such as enzymes, antibodies,hormones, and the like, the functional state of organs contributingthese factors can be monitored.

Accordingly, it will be seen that the present invention provides amethod for evaluation of hazardous agents in both in vivo and in vitroapplications. Although the description above contains manyspecificities, these should not be construed as limiting the scope ofthe invention but as merely providing illustrations of some of thepresently preferred embodiments of this invention. Thus, the scope ofthe invention should be determined by the appended claims and theirlegal equivalents.

What is claimed is:
 1. A method for evaluating the presence of an agent which is known or shown to activate or inhibit an enzyme present in human or animal saliva, comprising the steps of: providing a test saliva sample; analyzing activity of an enzyme in said test saliva sample; comparing said activity of said enzyme in said test saliva sample to a baseline activity of said enzyme determined from at least one control saliva sample that has not been exposed to an agent to establish if an agent is present in said test saliva sample; and determining the identity of said agent that is activating or inhibiting said enzyme in said test saliva sample.
 2. The method for evaluating the presence of an agent according to claim 1, wherein said analyzing step further comprises the steps of measuring a concentration of protein in said saliva sample and comparing and contrasting said measurement with a measurement of protein concentration in the control saliva sample.
 3. The method for evaluating the presence of an agent in saliva according to claim 1, wherein said analyzing step comprises the steps of: adding reactive substrate to said test saliva sample, said substrate specific for said enzyme present in said test saliva sample; incubating said test saliva sample and said reactive substrate; and detecting and measuring an amount of reaction products from said substrate and said enzyme in said test saliva sample as a means of quantifying the enzyme activity.
 4. The method for evaluating the presence of an agent in saliva according to claim 3, wherein, after said detecting and measuring step, said method further comprises the steps of: adding said reactive substrate to said at least one control saliva sample; incubating said control saliva sample and said reactive substrate; measuring a baseline amount of said reaction products from said at least one control saliva sample and contrasting with results from said test saliva sample.
 5. A method for evaluating the presence of a chemical or biological agent in selected media wherein said agent is known or shown to activate or inhibit an enzyme present in human or animal saliva comprising the steps of: providing a test media sample; providing an indicator saliva sample; combining said test media sample with said indicator saliva sample to form a combined sample; analyzing activity of said enzyme in said combined sample; and comparing the activity of said enzyme in said combined sample to a baseline activity determined from at least one control media sample in the absence of said agent.
 6. The method for evaluating the presence of a chemical or biological agent in selected media wherein said agent is known or shown to activate or inhibit an enzyme present in human or animal saliva according to claim 5, wherein said analyzing step further comprises the step of measuring a concentration of total protein or said enzyme for said test saliva sample.
 7. The method for evaluating the presence of a chemical or biological agent in media according to claim 6, wherein said analyzing step further comprises the steps of: adding at least one reactive substrate to said combined sample, said substrate specific for said saliva enzyme present in said indicator saliva sample; incubating said combined sample and said reactive substrate; and detecting and measuring the concentration in said combined sample of reaction product from said substrate and said saliva enzyme.
 8. The method for evaluating the presence of a chemical or biological agent in media according to claim 7, wherein said analyzing step further comprises the step of extracting and concentrating said test media sample before said combining.
 9. The method for evaluating the presence of a chemical or biological agent in media according to claim 8, wherein said test media sample is acquired from specimens from a group consisting of milk, urine, feces, blood, plasma, body tissue, water, air, and soil.
 10. A method for evaluating the exposure to an agent which is known or shown to activate or inhibit an enzyme present in human or animal saliva, comprising the steps of: providing a test saliva sample collected after said exposure; adding to said saliva sample a reactive substrate that is specific for said enzyme; detecting and measuring a reaction between said reactive substrate and said enzyme in said test saliva sample, wherein said reaction is increased or decreased by the presence of said agent; and determining exposure to said agent from the observed increase or decrease in said reaction in contrast to a baseline reaction level determined from at least one control saliva sample in the absence of said agent.
 11. A method for evaluating the presence of a chemical or biological agent in media, wherein said agent is known or shown to activate or inhibit an enzyme present in human or animal saliva, comprising the steps of: providing a test media sample that is suspected of containing said agent; providing an indicator saliva sample as a source of said enzyme; combining said test media sample with said indicator saliva sample to form a combined sample; adding a reactive substrate that is specific for said enzyme found in said indicator saliva sample to said combined sample; detecting and measuring a reaction between said enzyme in said combined sample said reactive substrate, wherein said reaction is increased or decreased by the presence of said agent; and determining the presence of said agent in said test media sample from the observed increase or decrease in said reaction compared to a baseline value obtained from at least one control media sample in the absence of said agent.
 12. A method for evaluating the exposure to a chemical agent, comprising the steps of: providing a test saliva sample from a test subject; adding to said provided test saliva sample a reactive substrate that is specific for a biomarker enzyme, said biomarker enzyme selected from the group consisting of acetyl cholinesterase, cholinesterase, alkaline phosphatase, amylase, carboxpeptidase, carboxylase, and esterase; incubating said test saliva sample and said reactive substrate; detecting and measuring a quantity of reactive product formed by a reaction between said biomarker enzyme and said substrate; and determining exposure to said agent by contrasting said detected and measured quantity of said reaction product in said test saliva sample with those of at least one control saliva sample, in the absence of said chemical agent, calculating an activity of said biomarker enzyme from said quantity of reaction product formation, and determining an activation or inhibition of said activity of said biomarker enzyme present in said test saliva sample, wherein said activation or inhibition of said activity of said biomarker enzyme is indicative of a presence of said chemical agent in said test saliva sample.
 13. A method for evaluating the presence of a chemical agent comprising the steps of: providing an indicator saliva sample containing a biomarker enzyme selected from the group consisting of acetylcholinesterase, cholinesterase, alkaline phosphatase, acid phosphatase, amylase, carboxpeptidase, carboxylase, and esterase; providing a test media sample containing said chemical agent; combining the test media sample containing said chemical agent with said indicator saliva sample; adding to said combined sample a reactive substrate specific for said biomarker enzyme; incubating said combined sample and said reactive substrate; detecting and measuring a concentration of a reactive product formed by a reaction between said biomarker enzyme and said substrate; and contrasting a detected concentration of said reaction product in said combined sample with those of at least one control combined media sample, free of said detected chemical agent, calculating activity of said biomarker enzyme from said detected and measured concentration of reaction product, and determining an inhibition of said activity of said biomarker enzyme present in said combined samples, wherein said inhibition of activity of said biomarker enzyme relative to control activity is indicative of a presence of said chemical agent in said test media sample.
 14. The method for evaluating the presence of a chemical agent according to claim 13, further comprising the step of extracting and concentrating said media sample containing said chemical agent before combining with said indicator saliva sample.
 15. The method for evaluating the presence of a chemical agent according to claim 14, further comprising the step of purifying said indicator saliva sample before combining with said media sample containing said chemical agent.
 16. The method for evaluating the presence of a chemical agent according to claim 15, further comprising the step of selecting said test media sample from a group consisting of milk, food substance, urine, feces, blood, plasma, body tissue, water, air, and soil.
 17. A method for evaluating the presence of an agent which is known or shown to activate or inhibit an enzyme present in human or animal saliva, comprising: providing a test saliva sample; analyzing activity of an enzyme in said test saliva sample; comparing said activity of said enzyme in said test saliva sample to a baseline activity of said enzyme determined from at least one control saliva sample that has not been exposed to an agent to establish if an agent is present in said test saliva sample; and correlating the observed activity of an enzyme with environmental exposure of a test subject to an agent which is known or shown to activate or inhibit an enzyme present in human or animal saliva.
 18. The method for evaluating the presence of an agent as recited in claim 17, further comprising: determining the identity of said agent that is activating or inhibiting said enzyme in said test saliva sample.
 19. A method for evaluating the presence of an agent which is known or shown to activate or inhibit an enzyme present in human or animal saliva, comprising: providing a test saliva sample; analyzing an amount of an enzyme in said test saliva sample; and comparing said amount of said enzyme in said test saliva sample to a baseline amount of said enzyme determined from at least one control saliva sample that has not been exposed to an agent to establish if an agent is present in said test saliva sample; and determining the identity of said agent that is present in said test saliva sample. 